This invention relates to antiviral agents, antimicrobial agents, and methods of use thereof. More particularly, illustrative embodiments of the invention relate to plasmids that encode antiviral agents that specifically destroy cells infected by human immunodeficiency viruses (“HIV”) that produce a protease in such infected cells. These antiviral agents are activated by the HIV protease, thereby specifically targeting HIV-infected cells for destruction. Other illustrative embodiments of the invention relate to plasmids that encode antimicrobial agents that specifically destroy cells infected by selected pathogenic microbes, such as Bacillus anthracis, the causal agent of anthrax.
Toxins that target cell surface receptors or antigens on tumor cells have attracted considerable attention for treatment of cancer. E.g., I. Pastan & D. FitzGerald, Recombinant Toxins for Cancer Treatment, 254 Science 1173-1177 (1991); Anderson et al., U.S. Pat. Nos. 5,169,933 and 5,135,736; Thorpe et al., U.S. Pat. No. 5,165,923; Jansen et al., U.S. Pat. No. 4,906,469; Frankel, U.S. Pat. No. 4,962,188; Uhr et al., U.S. Pat. No. 4,792,447; Masuho et al., U.S. Pat. Nos. 4,450,154 and 4,350,626. These agents include a cell-targeting moiety, such as an antigen-binding protein or a growth factor, linked to a plant or bacterial toxin. They kill cells by mechanisms different from conventional chemotherapy, thus potentially reducing or eliminating cross resistance to conventional chemotherapeutic agents.
Ricin and other similar plant toxins, such as abrin, modeccin and viscumin, comprise two polypeptide chains (known as the A and B chains) linked by a disulfide bridge, one chain (the A chain) being primarily responsible for the cytotoxicity and the other chain (the B chain) having sites that enable the molecule to bind to cell surfaces. Such toxins are known as type II ribosome-inactivating proteins or RIPs. F. Stirpe et al., Ribosome-inactivating Proteins from Plants: Present Status and Future Prospects, 10 Biotechnology 405-412 (1992).
Ricin is produced in the plant Ricinus communis (commonly known as the castor bean plant) via a precursor protein known as “preproricin.” Preproricin comprises a single polypeptide chain that includes a leader sequence, the A chain, a linker peptide, and the B chain. The leader sequence is subsequently removed in the organism to yield proricin, which is then cleaved to eliminate the linker region such that the A and B chains remain connected only by a disulfide bond in the mature protein. The toxicity of ricin-type toxins operates in three phases: (1) binding to the cell surface via the B chain; (2) penetration of at least the A chain into the cytosol via intracellular organelles, and (3) inhibition of protein synthesis through the A chain cleaving an essential adenine residue from ribosomal RNA. Thus, outside the cell separated A and B chains are essentially non-toxic, because the inherently toxic A chain lacks the ability to bind to cell surfaces and enter the cells in the absence of the B chain. Moreover, preproricin and proricin are also non-toxic, since the activity of the A chain is inhibited in these precursors.
It is also known that in ricin-type toxins the B chain binds to cell surfaces by virtue of galactose recognition sites, which react with glycoproteins or glycolipids exposed on the cell surface. It has been suggested that the toxicity of the ricin A chain might be exploited in anti-tumor therapy by replacing the indiscriminately-binding B chain with a different targeting component having the ability to bind only to tumor cells. Thus, various immunotoxins have been prepared consisting of a conjugate of whole ricin or a separated ricin A chain and a tumor-specific monoclonal antibody or other targeting component.
While previously described immunotoxins comprising ricin are generally suitable for their specific purposes, they possess certain inherent limitations that detract from their overall utility in treating viral or microbial infections. One problem with the known conjugates arises from a structural feature of the A chain from natural ricin. It is known that the natural ricin A chain becomes N-glycosylated during its synthesis, by enzymes present in Ricinus cells, and it is thought that the resulting sugar moieties are capable of non-specific interactions with cell surfaces. Thus, it appears that the known A chain conjugates are capable of a certain amount of binding with non target cells, even in the absence of the natural B chain, thus increasing the toxicity of such immunotoxins toward non-target cells. To partially mitigate this problem, recombinant A chain that lacks carbohydrate residues has been produced in E. coli. S. H. Pincus & V. V. Tolstikov, Anti-Human Immunodeficiency Virus Immunoconjugates, 32 Adv. Pharmacol. 205-247 (1995). Another problem with many ricin immunoconjugates arises from the fact that the B chain seems to have an important secondary function in that it somehow assists in the intoxication process, apart from its primary function in binding the ricin molecule to the cell surfaces. This secondary function is lost if the B chain is replaced by a different targeting component, such as a monoclonal antibody. Some researchers have addressed this problem by covalent attachment of affinity reagents to the B chain such that the galactose binding sites are blocked. J. M. Lambert et al., An Immunotoxin Prepared with Blocked Ricin: a Natural Plant Toxin Adapted for Therapeutic Use, 51 Cancer Res. 6236-6242 (1991).
The aforementioned modifications of ricin seek to enhance binding specificity to the outer cell surface by immunotoxins and similar, targeted therapeutic agents. Since certain types of infected cells do not express infection-related surface antigens, such binding specificity represents an inherent limitation. S. H. Pincus & V. V. Tolstikov, supra. A targeting-independent agent with a well-defined toxin activation mechanism involving a viral protease would permit the use of nonspecific “targeting” (i.e., cell-binding) molecules, including sugar moieties and fully active ricin B chain. Therapeutic agents designed in this manner could eliminate a broader spectrum of infected cells, with potentially fewer undesirable side effects.
Anti-HIV immunotoxins have been described that include antibodies linked to various toxic moieties via a peptide linker that includes a sequence cleavable by HIV protease. S. H. Pincus & V. V. Tolstikov, supra. In some cases, release of the toxic moiety by this protease may render it active, although the specific activation mechanism was not further defined.
U.S. Pat. No. 6,627,197 to W. K. Keener et al. describes antiviral toxins wherein an HIV protease cleavage site is interposed between ricin A and B chains. In ricin, the natural site for cleavage by proteolytic activity in Ricinus is in a disulfide-circumscribed loop in which one cysteine resides on the A chain and the other resides on the B chain; cleavage yields A and B chains connected by a disulfide bond. Therefore, U.S. Pat. No. 6,627,197 describes an HIV-protease cleavage sequence fused in-frame to the C-terminus of A chain such that the natural cleavage site is replaced with the HIV-protease site in the disulfide-circumscribed loop. In these embodiments, at lease some minimal N-terminal sequence of B chain required to inhibit A chain activity is retained, such that activation requires proteolytic cleavage and reduction of the disulfide bond.
As exemplified by U.S. Pat. No. 6,627,197, fusion proteins containing toxins have started to replace immunotoxins, which require chemical linking of the toxin to antibodies that bind cells. Such fusion proteins may contain portions of antibodies as targeting moieties. With an active toxin, the choice of targeting moieties is limited. Signal sequences, which are much shorter than antibodies, may promote translocation of agents into cells and increase the potency of the agents. Further, activatable toxins broaden the choices of targeting moieties. Further, conventional plasmid preparation is time consuming, thus restricting the number that can be tested. The development of highly versatile plasmids would accelerate the testing of signal sequences.
In view of the foregoing, it will be appreciated that providing highly versatile plasmids that permit the easy testing of signal sequences together with activatable toxins would be a significant advancement in the art.